Background. Although it is accepted that pediatric cancer treatment harbors a risk of gonadal damage, large cohort studies using up-to-date fertility markers are lacking. Procedure. The aim of our study was to evaluate the gonadal toxicity of childhood cancer treatment using fertility markers. We included 248 adult male long-term survivors of childhood cancer. Median age at diagnosis: 5 years, median age at follow-up: 24 years, median follow-up time 18 years. We evaluated patient characteristics, treatment modalities, testicular size, and endocrinological parameters including Inhibin B, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. Results. The median value of Inhibin B in the cancer survivor group was 126 ng/L versus 177 ng/L in the control group (P < 0.001). In the survivors, 67% had Inhibin B levels below the normal reference value of 150 ng/L compared with 26% in the control group (P < 0.05). Inhibin B was the most sensitive discriminator between survivors and controls. Significantly decreased Inhibin B levels and increased FSH levels were found in men treated for Hodgkin and non-Hodgkin lymphoma, acute-myeloid leukemia, neuroblastoma, and sarcoma as compared to other malignancies. Cumulative dosages of procarbazine and cyclophosphamide were the only independent chemotherapy-related predictors for decrease of Inhibin B levels and increase of FSH. Age at time of treatment did not influence post-treatment Inhibin B or FSH levels. Conclusions. Severe gonadal impairment is a risk in a considerable subgroup of childhood cancer survivors based on current fertility markers like Inhibin B. Males receiving gonadotoxic treatment before puberty are not protected from post treatment gonadal dysfunction.